Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration.
Ross et al BMC Cancer (2016) 16:553 DOI 10.1186/s12885-016-2570-0 RESEARCH ARTICLE Open Access PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma R L Ross1, H R McPherson1, L Kettlewell1, S D Shnyder2, C D Hurst1, O Alder1 and M A Knowles1* Abstract Background: Many urothelial carcinomas (UC) contain activating PIK3CA mutations In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases Methods: We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway Results: Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance Conclusions: Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer Keywords: PIK3CA, PI3K signaling, Bladder cancer, Urothelium Background Advanced urothelial carcinoma (UC) of the bladder has a poor prognosis At presentation, 15–30 % of UC patients are diagnosed with muscle-invasive tumors, and these carry a 5-year risk of death ranging from 33 to 73 % [1] The standard treatment for localized invasive UC, is surgical removal of the bladder and regional lymph nodes, but metastatic disease is a major cause of death in these patients The addition of cisplatincontaining combination neoadjuvant therapy has been shown to improve outcomes following cystectomy [2, 3], but metastasis remains common and although treatment with cisplatin-containing chemotherapy is beneficial in * Correspondence: m.a.knowles@leeds.ac.uk Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James’s University Hospital, Beckett Street, Leeds LS9 7TF, UK Full list of author information is available at the end of the article some cases, median survival for metastatic UC is only 13–15 months As no significant improvements in survival have been achieved in recent years, new approaches to therapy, particularly second line therapies for metastatic disease, are urgently needed Detailed molecular information on UC is now available [4, 5], but targeted agents have not yet been widely applied [6] The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in regulation of cell metabolism, migration, proliferation and survival [7] and mutations that lead to aberrant activation of the pathway are found in virtually all types of cancer In bladder cancer, 50–70 % of tumors contain mutations that are predicted to activate this pathway These include activating mutations in PIK3CA, [8, 9] and AKT1 [10], and inactivating mutations of PTEN [11, 12], PIK3R1 [13], TSC1 and TSC2 [9, 14] Assessment of the phosphorylation status of key pathway © 2016 The Author(s) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Ross et al BMC Cancer (2016) 16:553 proteins confirms that pathway activation is present in bladder tumors of all grades and stages [15] These tumors may benefit from PI3K-targeted therapy Clinical trials of mTORC1 inhibitors in patients with bladder cancer have been initiated in recent years In trials of the mTOR inhibitor Evirolimus, exceptional responses have been reported in patients with advanced UC whose tumors contained TSC1 or mTOR mutations [16, 17] In general however, responses to mTOR inhibitors have not been impressive [18], and indeed not all UC patients with tumors containing TSC1 mutations have shown responses [16] A potential reason is that mTOR inhibition triggers feedback loops that activate AKT [19] Inhibitors of AKT have therefore been examined in preclinical studies of UC [20, 21] Importantly, these studies revealed that sensitivity to AKT inhibition was strongly related to the presence of PIK3CA mutation Taken together, it is clear that a thorough understanding of the signaling events initiated by the PI3K pathway is required in order to maximize clinical benefit Inhibition of PI3K as a potential therapeutic approach in UC has not previously been examined, though mutations in PIK3CA represent the most frequent PI3K pathway mutations in this cancer type, including 12–20 % of muscle-invasive tumors [14, 22] Preclinical studies and early clinical trials indicate sensitivity to inhibitors of PI3K in several cancers including breast, ovarian, endometrial, lung and multiple myeloma [18, 23–29] The majority of these studies highlight the Class PI3K inhibitor, GDC-0941, as a good therapeutic drug for solid tumors Furthermore, a phase I dose-escalation study of GDC-0941 has recently been completed and reports good tolerability of the drug with confirmed target modulation in tumor tissues [30] Several studies in non-bladder cell lines have sought predictive biomarkers of sensitivity to PI3K inhibitors and it has been suggested that mutation of PIK3CA or loss of PTEN function are related to sensitivity to inhibitors of class I PI3K and that mutations in RAS genes are associated with resistance (Reviewed in [31]), though prediction based on these biomarkers is not absolute Previously we examined the effect of ectopic expression of mutant PIK3CA in telomerase-immortalized normal human urothelial cells (TERT-NHUC) and showed that this induces cell proliferation and migration [32] In bladder tumors, more than one lesion in the PI3K pathway is commonly present [9] and this could potentially lead to distinct types of pathway dependence and response to specific therapeutic agents Therefore, we have examined the consequences of specific inhibition of mutant PIK3CA in UC cells using stable knockdown, and treatment of a panel of UC cell lines containing a range of PI3K pathway alterations with the class I PI3K inhibitor, GDC-0941 Our findings strongly suggest that targeting of PIK3CA maybe a valid therapeutic approach in advanced bladder cancer Page of 12 Methods Cell culture Cell lines with known PI3K pathway mutation status were chosen (Additional file 1) Cell lines used for gene knockdown and functional studies were VM-CUB-3, BFTC909 and 253J VM-CUB-3 was established from a primary human bladder transitional cell carcinoma (TCC), the grade and stage of which are unknown [33] BFTC909 was established from the sarcomatoid component of a grade TCC of the renal pelvis [34] 253J was established from a retroperitoneal metastasis from a human TCC [35] Bladder cancer cell lines J82, 253J, HT-1197, VMCUB-3, BFTC909, UM-UC3, KU-19-19, DSH1, VM-CUB1, CAL29, TCCSUP, MGH-U3, 639V, 97-1, LUCC1, LUCC3 and RT4 were used in drug sensitivity assays Cell line identity was verified by short tandem repeat DNA typing using the Powerplex 16 kit (Promega) Profiles were compared to publically available data (ATCC, DSMZ) or where no reference profile was available, were confirmed as unique Cells were grown in standard growth media; Hams F12 + % FCS + % Insulin-Transferrin-Selenium + μg/ml hydrocortisone + 1x Non-essential amino acids + mM L-glutamine (97-1), MEM + 10 % FCS + 1x Nonessential amino acids + mM L-glutamine (HT-1197, J82, MGH-U3), DMEM + 10 % FCS + mM L-glutamine (VM-CUB-3, VM-CUB-1, TCCSUP, BFTC909, 639V, CAL29, UM-UC3), McCoy’s 5a + 10 % FCS + mM L-glutamine (RT4), 50:50 DMEM and RMPI 1640 + % FCS + mM L-glutamine (253J) and RPMI 1640 + 10 % FCS + mM L-glutamine (DSH1, KU-19-19) Cells were incubated at 37 °C in % CO2 TERT-NHUC [36] were also used and were cultured in Keratinocyte Growth Medium Kit plus supplements (with 90 μl CaCl2) All cells were tested routinely for mycoplasma whilst in culture and before freezing by PCR using PCR Mycoplasma Test Kit I/C (PromoKine PK-CA91-1048) according to the manufacturer’s protocol shRNA constructs and transduction of cell lines Two shRNAs targeting PIK3CA were designed (forward oligo 5′- gcagaagtatactctgaaatTCAAGAGatttcagagtatac ttctgcTTTTTTGGGCC-3′, reverse oligo 5′- CAAA AAAgcagaagtatactctgaaatCTCTTGAatttcagagtatacttctgc -3′, forward oligo 5′- caggtatctaccatggaggtTCAAGA GacctccatggtagatacctgTTTTTTGGGCC-3′ and reverse oligo 5′- CAAAAAAcaggtatctaccatggaggtCTCTTGA acctccatggtagatacctg-3′ according to an algorithm described previously [37, 38] The sequence in lowercase is complementary to PIK3CA and forms a short-hairpin structure when expressed due to the intervening loop sequence These, and a non-specific (NS) shRNA, were first cloned into pGEM-U6 puro and then into pRetroSuperpuro (pRS-puro), along with empty pRS-puro vector Ross et al BMC Cancer (2016) 16:553 (control), to generate retroviruses to transduce VM-CUB-3, BFTC909 and 253J cell lines as described previously [37] Western blotting Protein extraction was carried out as described [39] and concentration was quantified using the BIO-RAD protein assay (BIO-RAD, Hemel Hempstead, UK) SDSPAGE and immunoblotting was carried out as described [32] Primary antibodies were anti-p110a, anti-pAKT (Ser473), anti-panAKT (Cell Signaling) and anti-tubulin alpha (AbD Serotec) Bound primary antibodies were detected using HRP-conjugated secondary antibodies and Luminata Forte Western HRP Substrate (Millipore) Phenotypic assays Proliferation, anchorage-independent growth, and Transwell migration assays were carried out and analyzed as described previously [32] All assays were done in triplicate and repeated at least three times Xenografts Xenografts were established in mice by subcutaneous inoculation of VM-CUB-3 cells with PIK3CA knockdown and control cells (NS shRNA knockdown) Pure strain male BALB/cOlaHsd-Fox1nu mice aged to weeks were used as described [40] Each cell line was injected subcutaneously into sites on both flanks of mice at a concentration of × 107 cells/site On day following inoculation, tumor was evident, and tumor volume was then measured frequently using calipers ((a2xb)/2; where a is the smaller and b is the larger diameter of the tumor) up to day 43 Tumor volume is shown in mm3 Immunohistochemistry Tumors were formalin-fixed and embedded in paraffin wax Sections were stained with haematoxylin and eosin, anti-human Ki-67 proliferation-associated antibody (Dako) and for apoptosis using the terminal deoxynucleotidyl transferase–mediated dUTP nick-end labelling (TUNEL) assay (ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit; Chemicon) and analyzed as described previously [40] GDC-0941 drug treatment The class I PI3K inhibitor, GDC-0941 (Axon Medchem), was used to treat bladder cancer cell lines and TERTNHUC The dose range chosen was based on previous studies that report IC50 values of 0.28–0.95 μM for cell viability of solid tumor cell lines, as well as pharmacokinetic data available from phase I clinical studies that report a maximum of μM GDC-0941 plasma concentration in patients [30, 41] Cell viability was assessed by CellTiter-Blue® (Promega) analysis of bladder cancer cell lines and TERT-NHUC subjected to GDC-concentrations Page of 12 from to μM Cell viability was assessed by CellTiterBlue® (Promega) analysis 1000–4000 cells per well (number of cells determined to ensure that confluence was not achieved in untreated controls during the experiment) were plated in 96-well plates in five replicate wells and allowed to attach for 24 h before addition of 0–2 μM GDC-0941 in 0.1 % DMSO After 72 h, 20 μl of CellTiterBlue solution was added to the medium for h and fluorescence read at 550 nm Medium alone was used as a blank Prism software (GraphPad Software, La Jolla, CA, USE) was used to calculate IC50 values Cell cycle and apoptosis analysis of cells cultured with μM GDC-0941 or DMSO only for 48 h was evaluated by flow cytometry as described [40] All assays were done in triplicate and repeated at least three times Statistical analysis Tumor growth was analysed using the Mann-Whitney Utest Drug IC50 data was analyzed using the Fisher exact test (two-tailed, based on a cut off of >1 and 50 μm in an area of 2.5 mm2 after weeks of incubation in soft agarose c Mean fluorescence of migrated DAPIstained VM-CUB-3 cells migrating through a Transwell membrane towards a chemoattractant (serum-supplemented medium) Values represent mean ± S.E Results are representative of triplicate experiments * indicates statistical significant difference (ANOVA test) Values represent mean ± S.E puromycin-resistant cell populations, p110α protein levels (Fig 3a) and AKT activation (data not shown) were confirmed as described above Cells were injected subcutaneously Tumor volume (mm3) was measured from day to day 43 p110α-KD xenografts showed significantly reduced growth rate compared with controls (Mann-Whitney test, P < 0.05) (Fig 3b) Student t test showed that by day 42, the difference between tumor volume of KD and control xenografts was significantly different (Adjusted P < 0.017; on day 43 P < 0.005) No significant differences were identified in tumor histology (data not shown) Sections were stained for Ki67 and TUNEL to assess effects on proliferation and apoptosis, respectively A significant decrease in the proliferative index of p110α-KD tumors was found (ANOVA test, P < 0.05) (Fig 3c and Additional file 3) but significant numbers of apoptotic cells were not detected, consistent with in vitro data Effects of GDC-0941 on cell viability The effects of mutant PIK3CA knockdown in UC cells suggest that this is a major driver of transformation in this cell type and that mutant PIK3CA is a good candidate for therapeutic targeting in UC Therefore we assessed the effect of GDC-0941, a small molecule ATPcompetitive inhibitor of class IA PI3K isoforms (α, β, δ), on a panel of UC cell lines and TERT-NHUC We selected a panel of 17 UC cell lines with known mutation status for PIK3CA, PIK3R1, AKT1, TSC1, PTEN and the three RAS genes [9, 10, 13, 42] (Additional file 1) RNA expression data for PIK3CA, PIK3CB and PIK3CD were available for these and for normal human urothelial cells (Hurst and Knowles, unpublished data) In 13 of 17 lines, expression of PIK3CA was >2-fold higher than in normal urothelial cells Six of these also showed >2-fold upregulation of PIK3CB Expression of PIK3CD was >2fold lower than in normal urothelial cells in all but two cell lines (Additional file 4) Cells were cultured in 0-2.0 μM GDC-0941 for 72 h and cell viability measured relative to untreated controls Sensitivity values (IC50) to this compound were within the same range as seen in other cancer cell types [25, 44] Eight of the 10 cell lines with mutant PIK3CA showed IC50 values from 0.4 μM to 1.25 μM (Fig and Additional file 5) Six of these eight cell lines have only PIK3CA mutation and harbor additional PIK3R1 (LUCC3) or NRAS Q61R (HT-1197) mutations Overall cell lines with wild-type PIK3CA were significantly less sensitive to GDC-0941 treatment than mutant PIK3CA cell lines (Fisher exact test, P < 0.05; two-tailed based on a cut off of >1 and